Reconstitution of cellulose and lignin after [C2mim][OAc] pretreatment and its relation to enzymatic hydrolysis

Although the effects of cellulose crystallinity and lignin content as two major structural features on enzymatic hydrolysis have been extensively studied, debates regarding their effects still exist. In this study, reconstitution of cellulose and lignin after 1‐ethyl‐3‐methylimidazolium acetate ([C₂mim][OAc]) pretreatment was proposed as a new method to study their effects on enzymatic digestibility. Different mechanisms of lignin content for reduction of cellulose hydrolysis were found between the proposed method and the traditional method (mixing of cellulose and lignin). The results indicated that a slight change of the crystallinity of the reconstituted materials may play a minor role in the change of enzyme efficiency. In addition, the present study suggested that the lignin content does not significantly affect the digestibility of cellulose, whereas the conversion of cellulose fibers from the cellulose I to the cellulose II crystal phase plays an important role when an ionic liquid pretreatment of biomass was conducted. Biotechnol. Bioeng. 2013; 110: 729–736. © 2012 Wiley Periodicals, Inc.     

Thermogravimetry study of xylanase- and laccase/mediator-treated eucalyptus pulp fibres

Thermogravimetric analyses (TGA) was applied to study the effects of enzymatic bleaching of eucalyptus pulp with xylanase and a laccase-mediator system. The thermal degradation profile of the pulps was sensitive to the enzymatic treatments. Xylanase treatment produced an ordered and clean microfibril, whereas laccase oxidized surface cellulose chains and increased the amorphous (paracrystalline) cellulose content. In this case, pulp viscosity decreased from 972 to 859 mL/g and apparent pulp crystallinity calculated from TGA data decreased almost 50%. Alkaline extraction was necessary to recover pulp crystallinity and to remove oxidized lignin in the laccase-treated samples. TGA data allowed differentiating and quantifying crystalline and amorphous cellulose. This thermogravimetric approach is a simple method in order to monitor superficial changes in cellulosic microfibrils.     

Effect of drying and rewetting cycles on the structure and physicochemical characteristics of softwood fibres for reinforcement of cementitious composites

The changes produced in cellulosic fibres when they are subjected to successive drying and rewetting cycles could have an important impact on the resistance and durability of cement mortar composites based on these fibres. In this paper, unbleached, oxygen delignificated, semi-bleached, and fully bleached softwood pulps have been subjected to drying and rewetting cycles and the corresponding dried pulps characterized. The morphological structures and thermal stabilities were investigated with X-ray diffraction and thermogravimetric analysis. While the water retention values decrease significantly with drying and rewetting cycles, an overall increase in the crystallinity index and in the thermal stability was found in the hornificated pulps. Natural fibres from cotton linters were also studied and the results compared with the fibres from these softwood pulps.     

Cellulase digestibility of pretreated biomass is limited by cellulose accessibility

Attempts to correlate the physical and chemical properties of biomass to its susceptibility to enzyme digestion are often inconclusive or contradictory depending on variables such as the type of substrate, the pretreatment conditions and measurement techniques. In this study, we present a direct method for measuring the key factors governing cellulose digestibility in a biomass sample by directly probing cellulase binding and activity using a purified cellobiohydrolase (Cel7A) from Trichoderma reesei. Fluorescence-labeled T. reesei Cel7A was used to assay pretreated corn stover samples and pure cellulosic substrates to identify barriers to accessibility by this important component of cellulase preparations. The results showed cellulose conversion improved when T. reesei Cel7A bound in higher concentrations, indicating that the enzyme had greater access to the substrate. Factors such as the pretreatment severity, drying after pretreatment, and cellulose crystallinity were found to directly impact enzyme accessibility. This study provides direct evidence to support the notion that the best pretreatment schemes for rendering biomass more digestible to cellobiohydrolase enzymes are those that improve access to the cellulose in biomass cell walls, as well as those able to reduce the crystallinity of cell wall cellulose.     

Cellulase-assisted refining of chemical pulps: Impact of enzymatic charge and refining intensity on energy consumption and pulp quality

Paper production requires fibres to be refined, meaning mechanically treated to present sufficient bonding potential. As it is a highly energy consuming stage, various charges of cellulase as a pre-treatment were investigated to reduce the energy consumption and improve paper properties. The enzyme was added during pulp slushing and conditions of treatment were chosen to be compatible with an industrial application. Results obtained after low consistency disc refining of a softwood bleached kraft pulp were compared at a given drainage index. Enzymatically-treated samples showed a better development of fibrillation leading to a stronger paper. Moreover, fibre swelling was significantly improved. The impact of cellulase charge added to the pulp was identified. However, for all cellulase charges tested, fibre intrinsic resistance was lowered. Consequently fibres became shorter and tear index dropped. A solution was found by applying a milder treatment consisting in reducing refining intensity. This was a way to limit the intense fibre cutting at high refining levels. Moreover, by treating pulp with cellulase, it became possible to reduce refining intensity by 33%, keeping breaking length similar to control.     

Effects of organosolv pretreatment and enzymatic hydrolysis on cellulose structure and crystallinity in Loblolly pine

Ethanol organosolv pretreatment was performed on Loblolly pine to enhance the efficiency of enzymatic hydrolysis of cellulose to glucose. Solid-state ¹³C NMR spectroscopy coupled with line shape analysis was used to determine the structure and crystallinity of cellulose isolated from pretreated and enzyme-hydrolyzed Loblolly pine. The results indicate reduced crystallinity of the cellulose following the organosolv pretreatment, which renders the substrate easily hydrolyzable by cellulase. The degree of crystallinity increases and the relative proportion of para-crystalline and amorphous cellulose decreases after enzymatic hydrolysis, indicating preferential hydrolysis of these regions by cellulase. The structural and compositional changes in this material resulting from the organosolv pretreatment and cellulase enzyme hydrolysis of the pretreated wood were studied with solid-state CP/MAS ¹³C NMR spectroscopy. NMR spectra of the solid material before and after the treatments show that hemicelluloses and lignin are degraded during the organosolv pretreatment.     

Effect of beating on recycled properties of unbleached eucalyptus cellulose fiber

The effects of beating on recycled properties of eucalyptus cellulose fiber were studied by analyzing the changes of morphological parameters (fiber length and the fines content), physical properties (tensile strength, breaking length and the stretch), WRV, crystal structure of cellulose and pore structure of cellulose fiber. The results showed that beating caused the fine content increase. Tensile strength, breaking length and the stretch increased with the increasing beating time. WRV of the first cycle beaten eucalyptus pulp was increased by 32.1%, compared to the first cycle unbeaten pulp. WRV increased with the increase in beating degree. However, crystallinity of cellulose increased, and then decreased with an increase in beating degree. FTIR spectra showed that there were no drastic changes in the functional groups of the eucalyptus pulp cellulose during beating. Fiber pore size was gradually diverted into macropore with the increase in beating degree, resulting in the mean pore volume increased.     

Surface character of pulp fibres studied using endoglucanases

The endoglucanase Cel5A from Trichoderma reesei and an endoglucanase from Aspergillus sp. (Novozym 476 from Novozyme A/S) were evaluated as probes for the surface properties of soft- and hardwood chemical pulp fibres. The hydrolysis time curves were in accordance with a two-phase degradation model described by a biexponential function. The kinetic parameters corresponding to the amount of fast and slow degraded parts of the substrate correlated to tensile index, relative bonded area and z-strength of the paper. All paper properties showing a correlation with enzyme kinetic parameters were related to fibre-fibre interactions. Fluorescence labelling of the reducing end groups in pulp fibres followed by enzyme treatment indicated that the fast substrate class corresponds to the population of "loose" cellulose chain ends not tightly associated with the bulk cellulose. The correlation between the parameters of enzyme kinetics and mechanical properties of the paper produced from the corresponding pulp found in this study should allow a rapid evaluation of the raw fibre material used in paper making process.     

Correlating physical changes and enhanced enzymatic saccharification of pine flour pretreated by N-methylmorpholine-N-oxide

Pretreatment of lignocellulosic biomass by N-methylmorpholine-N-oxide (NMMO), a solvent used in the textile industry to dissolve cellulose for production of regenerated cellulose fibers, was observed to enhance significantly enzymatic saccharification and fermentation. The enhancement was speculated to have been caused by reduced cellulose crystallinity after dissolution and precipitation processes. This work focused on assessing several physical changes and their correlations to enzymatic saccharification of pine flour after NMMO pretreatment. Results from microstructure, surface chemical composition, and cellulose accessibility complementarily illustrated the enrichment of cellulose on pine flour surface after NMMO pretreatment. Cellulose accessibility was highly correlated to the overall glucan conversion rate. Changes in crystallinity were correlated to the initial hydrolysis rate but not overall glucan conversion rate. Findings from this work may contribute to lignocellulosic bioenergy from development of novel pretreatment technologies utilizing NMMO.     

Analysis of retted and non retted flax fibres by chemical and enzymatic means

Flax fibres (Linum usitatissimum L.) were subjected to chemical and enzymatic analysis in order to determine the compositional changes brought about by the retting process and also to determine the accessibility of the fibre polymers to enzymatic treatment. Chemical analysis involved subjecting both retted and non retted fibres to a series of sequential chemical extractions with 1% ammonium oxalate, 0.05 M KOH, 1 M KOH and 4 M KOH. Retting was shown to cause minimal weight loss from the fibres but caused significant changes to the pectic polymers present. Retted fibres were shown to have significantly lower amounts of rhamnogalacturonan as well as arabinan and xylan. In addition the average molecular mass of the pectic extracts was considerably lowered. Enzyme treatment of the 1 M KOH extracts with two different enzymes demonstrated that the non retted extract contained a relatively high molecular weight xylan not found in the retted extract. Treatment of the 1 M KOH extracts and the fibres with Endoglucanase V from Trichoderma viride demonstrated that while this enzyme solubilised cellulose as well as xylan and xyloglucan oligomers from the extract, it had limited access to these polymers on the fibre. MALDI-TOF MS analysis of the material solubilised from the extract suggested that the xylan was randomly substituted with 4-O-methyl glucuronic acid moieties. The xyloglucan was shown to be of the XXXG type and was substituted with galactose and fucose units. The enzyme treatments of the fibres demonstrated that the xylan and xyloglucan polymers in the fibres were not accessible to the enzyme but that material which was entrapped by the cellulose could be released by the hydrolysis of this cellulose.     

Lignin-hemicellulose complexes restrict enzymatic solubilization of mannan and xylan from dissolving pulp

Mannan and xylan present in bleached softwood dissolving pulp were found to be partially resistant to hemicellulases even after repeated enzyme treatment. Despite the additional effect of an endoglucanase from Gloeophyllum sepiarium, which increased the␣accessibility of mannan and xylan to a mannanase from Sclerotium rolfsii and to a xylanase from Thermomyces lanuginosus, the enzyme mixture solubilized only half of the hemicellulose present in the pulp. Half of the remaining hemicellulose present in the pulp appeared to be entrapped within the cellulose matrix while the other half was associated with lignin-carbohydrate complexes. The latter hemicellulose portion was isolated and characterized. Chromatography and spectroscopic techniques revealed the presence of two types of lignin-carbohydrate complex, a galactoglucomannan-lignin complex (degree of polymerization DP 50–60) and a xylan-lignin complex (DP >200). Received: 8 December 1997 / Received revision: 30 April 1998 / Accepted: 8 May 1998     

Structural features affecting biomass enzymatic digestibility

The rate and extent of enzymatic hydrolysis of lignocellulosic biomass highly depend on enzyme loadings, hydrolysis periods, and structural features resulting from pretreatments. Furthermore, the influence of one structural feature on biomass digestibility varies with the changes in enzyme loading, hydrolysis period and other structural features as well. In this paper, the effects of lignin content, acetyl content, and biomass crystallinity on the 1-, 6-, and 72-h digestibilities with various enzyme loadings were investigated. To eliminate the cross effects among structural features, selective pretreatment techniques were employed to vary one particular structural feature during a pretreatment, while the other two structural features remained unchanged. The digestibility results showed that lignin content and biomass crystallinity dominated digestibility whereas acetyl content had a lesser effect. Lignin removal greatly enhanced the ultimate hydrolysis extent. Crystallinity reduction, however, tremendously increased the initial hydrolysis rate and reduced the hydrolysis time or the amount of enzyme required to attain high digestibility. To some extent, the effects of structural features on digestibility were interrelated. At short hydrolysis periods, lignin content was not important to digestibility when crystallinity was low. Similarly, at long hydrolysis periods, crystallinity was not important to digestibility when lignin content was low.     

Activity of laccase on unbleached and bleached thermomechanical pulp

The introduction of value-added properties to pulp fibres is an attractive proposition. One interesting option is targeted modification of fibre surfaces by enzymatic activation. In this work, the activity of laccase on pulp and pulp fractions from thermomechanical pulp (TMP) and peroxide bleached TMP was studied on the basis of consumption of the co-substrate oxygen in the reaction and by studying the formation of radicals in the pulp material as analysed by electron paramagnetic resonance spectroscopy (EPR). Laccases obtained from Trametes hirsuta and Myceliophthora thermophila were used in the study. Laccases were found to be active on pulp material of unbleached TMP, whereas only fines from bleached TMP reacted in laccase treatment. Dissolved and colloidal substances (DCS) were assumed to have a mediating role in the laccase-catalysed oxidation of the fibre-bound material.     

Ca(2+) sorption on regenerated cellulose fibres

High calcium content in cellulose materials can cause considerable problems in pulp processing, textile chemical treatment and consumer use, e.g. dyeing operations or household laundry. The Ca(2+) binding capacity of cellulose also is of relevance in food and medical applications. Through their carboxyl group content regenerated cellulose fibres can act as weak anion exchangers, thus all types of regenerated cellulose fibres such as lyocell, viscose and modal fibres, show a distinct ability to bind Ca(2+) ions. The binding capacity is limited by the carboxyl group content, which was determined with 15mmol/kg for lyocell fibres and 20mmol/kg for viscose fibres, using the Methylene Blue sorption method. The presence of bound Ca(2+) also was demonstrated by complex formation with alizarin. The molar ratio between carboxylic group content and bound Ca(2+) ions was one Ca(2+) ion for a single carboxyl group. As a result of Ca(2+) sorption a positive net charge of the cellulose results and another anion has to be bound as counter ion for reasons of charge neutralisation. Results of potentiometric titrations indicate HCO(3)(-) to be present as counter ion in the Ca(2+) cellulose system. Thus under the experimental conditions studied, bound Ca(2+) is proposed to be present in the form COO(-)Ca(2+)HCO(3)(-).     

Homogeneous suspensions of individualized microfibrils from TEMPO-catalyzed oxidation of native cellulose

Never-dried native celluloses (bleached sulfite wood pulp, cotton, tunicin, and bacterial cellulose) were disintegrated into individual microfibrils after oxidation mediated by the 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) radical followed by a homogenizing mechanical treatment. When oxidized with 3.6 mmol of NaClO per gram of cellulose, almost the totality of sulfite wood pulp and cotton were readily disintegrated into long individual microfibrils by a treatment with a Waring Blendor, yielding transparent and highly viscous suspensions. When observed by transmission electron microscopy, the wood pulp and cotton microfibrils exhibited a regular width of 3-5 nm. Tunicin and bacterial cellulose could be disintegrated by sonication. A bulk degree of oxidation of about 0.2 per one anhydroglucose unit of cellulose was necessary for a smooth disintegration of sulfite wood pulp, whereas only small amounts of independent microfibrils were obtained at lower oxidation levels. This limiting degree of oxidation decreased in the following order: sulfite wood pulp > cotton > bacterial cellulose, tunicin.     

Influence of the cycle number in processing of cellulose from waste paper on its ability to hydrolysis by cellulases

Hydrolytic ability of laboratory enzyme preparations from fungus of the Penicillium genus was investigated using kraft pulp from nonbleached softwood and bleached hardwood cellulose as substrates. The enzyme preparations were shown to efficiently hydrolyze both softwood and hardwood cellulose. The yields of glucose and reducing sugars were 24–36 g/l and 27–37 g/l from 100 g/l of dry substrate in 48 h, respectively, and depended on the number of substrate grinding cycles.     

Characterization of the crystalline structure of cellulose using static and dynamic FT-IR spectroscopy

The cellulose structure is a factor of major importance for the strength properties of wood pulp fibers. The ability to characterize small differences in the crystalline structures of cellulose from fibers of different origins is thus highly important. In this work, dynamic FT-IR spectroscopy has been further explored as a method sensitive to cellulose structure variations. Using a model system of two different celluloses, the relation between spectral information and the relative cellulose Ialpha content was investigated. This relation was then used to determine the relative cellulose Ialpha content in different pulps. The estimated cellulose I allomorph compositions were found to be reasonable for both unbleached and bleached chemical pulps. In addition, it was found that the dynamic FT-IR spectroscopy technique had the potential to indicate possible correlation field splitting peaks of cellulose Ibeta.     

Fractionation of sugar beet pulp into pectin, cellulose, and arabinose by arabinases combined with ultrafiltration.

Incubation of beet pulp with two arabinases (alpha-L-arabinofuranosidase and endo-arabinase), used singularly or in combination at different units of activity per gram of beet pulp, caused the hydrolysis of arabinan, which produced a hydrolyzate consisting mainly of arabinose. Pectin and a residue enriched with cellulose were subsequently separated from the incubation mixture. The best enzymatic hydrolysis results were obtained when 100 U/g of beet pulp of each enzyme worked synergistically with yields of 100% arabinose and 91.7% pectin. These yields were higher than those obtained with traditional chemical hydrolysis. The pectin fraction showed a low content of neutral sugar content and the cellulose residue contained only a small amount of pentoses. Semicontinuous hydrolysis with enzyme recycling in an ultrafiltration unit was also carried out to separate arabinose, pectin, and cellulose from beet pulp in 7 cycles of hydrolysis followed by ultrafiltration. The yields of separation were similar to those obtained in batch experiments, with an enzyme consumption reduced by 3.5 times and some significant advantages over batch processes.     

TCF bleaching of wheat straw pulp using ozone and xylanase. Part A: paper quality assessment

The XOAZRP TCF sequence was applied to bleach wheat straw pulp. Following each bleaching stage, the properties of the pulp (viz. kappa number, standard viscosity, borohydride viscosity and brightness) and of the resulting effluents were determined. The performance of the reagents was analyzed through the studies of xylanase treatment and crystallinity and scanning electron microscopy of the pulps. Finally, the pulp was refined at 1000 revolutions in a PFI mill and the mechanical properties of the resulting paper were determined and compared with those of paper from a eucalyptus pulp. Despite its shortcomings, wheat straw pulp can be effectively bleached with the proposed TCF sequence.     

Pectinase in papermaking: solving retention problems in mechanical pulps bleached with hydrogen peroxide

Treatment with the enzyme pectinase has been reported to lower the cationic demand of thermomechanical pulp (TMP) bleached with alkaline peroxide in the laboratory. We have extended this discovery to bleached TMP produced industrially, and shown that commercial enzyme preparations can treat pulp within 15 min at the temperature and pH values prevalent in paper mills. About half of the cationic demand in the bleached pulp can be destroyed by pectinase. Dynamic drainage jar experiments show that the enzyme treatment improves the effectiveness of several cationic polymers to increase retention of fines and filler particles. It does not increase retention in the absence of retention aids or with nonionic polymers, and does not damage the strength properties of the pulp. Pectinase could be easily incorporated into paper machine stock preparation systems to lower the charges of cationic retention aids needed in furnishes containing peroxide-bleached mechanical pulp.